This disclosure relates to devices and methods for sample processing and characterization, and various uses thereof. In particular, this disclosure relates to devices and methods for separation and characterization of analytes in a mixture of analytes.
Separation of analyte components from a more complex analyte mixture on the basis of one or more inherent qualities of the analytes, and optionally providing sets of sample fractions that are enriched for specific analyte components, is a key part of analytical chemistry. Simplifying complex mixtures in this manner reduces the complexity of downstream analysis. In some cases, it can be advantageous to perform two or more enrichment steps that are orthogonal (e.g., based on different and/or unrelated qualities). In many cases, however, the process of performing orthogonal enrichment steps using known methods and/or devices is cumbersome, and can dilute the analyte to a concentration that is beyond the detection sensitivity of the downstream analytical equipment. In addition, complications can arise when attempting to interface known enrichment methods and/or devices with analytical equipment and/or techniques. In some instances, sample separation and/or enrichment may be performed upstream or in parallel with sample analysis. For example, devices for performing sample enrichment may be coupled directly with an analytical instrument.
A variety of methods have been used, for example, to interface protein sample preparation techniques with downstream detection systems such as mass spectrometers. A common method is to prepare samples using liquid chromatography and collect fractions for mass spectrometry. This has the disadvantage of requiring protein samples to be separated into a large number of sample fractions which must be analyzed, and complex data reconstruction must be performed post-run. While certain forms of liquid chromatography can be coupled to a mass spectrometer (LC-MS), for example peptide map reversed-phase chromatography, these known techniques are restricted to using peptide fragments, rather than intact proteins, which limits their utility.
Another method to introduce samples into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of sample and solution at a distal end of a capillary or microfluidic device are ionized to induce an attraction to the charged plate of a mass spectrometer. The droplet then stretches in this induced electric field to a cone shape (“Taylor cone”), which then releases small droplets into the mass spectrometer for analysis. Typically this is done in a capillary, which provides a convenient volume and size for ESI. Capillaries however, provide a linear flow path that does not allow for multi-step processing.
Other work has been pursued with microfluidic devices. Microfluidic devices may be produced by various known techniques and provide fluidic channels of defined dimensions that can make up a channel network designed to perform different fluid manipulations. These devices offer an additional level of control and complexity compared to capillaries, making them a better choice for sample prep. However, as with capillary-based systems, these tools often provide limited characterization of separated analyte fractions prior to introduction to a mass spectrometer.
One application for protein mass spectrometry is characterization of proteins during the development and manufacturing of biologic and biosimilar pharmaceuticals. Biologics and biosimilars are a class of drugs which include, for example, recombinant proteins, antibodies, live virus vaccines, human plasma-derived proteins, cell-based medicines, naturally-sourced proteins, antibody-drug conjugates, protein-drug conjugates and other protein drugs.
Regulatory compliance requires extensive testing of biologics during development and manufacture, something that is not required for small molecule drugs. This is because the manufacture of biologics has greater complexity due to, for example, using living material to produce the biologic, the greater complexity of the biologic molecule itself, and greater complexity of the manufacturing process. Characteristics required to be defined include, for example, mass, charge, changes in hydrophobicity, and glycosylation state, as well as efficacy. Currently these tests are performed independently of each other, leading to a very time consuming and expensive process for characterizing biologics.
Methods, devices, and systems for performing analyte separations, improving the accuracy of quantitative separation data, and achieving improved correlation between the quantitative separation data and downstream analytical characterization data, e.g., mass spectrometry characterization data, are described in the present disclosure.